Sustainable Chemistry and Pharmacy 6 (2017) 82–89

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Sustainable Chemistry and Pharmacy

journal homepage: www.elsevier.com/locate/scp

Perspectives for the use of biotechnology in green chemistry applied to

T
biopolymers, fuels and organic synthesis: from concepts to a critical point of
view
Maíra Fasciotti
Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO, Divisão de Metrologia Química e Térmica, Diretoria de Metrologia Científica e Industrial, 25250-
020, Xerém, Duque de Caxias, Brazil

ARTICLE INFO ABSTRACT

Keywords: Since the eighteenth century, after the first industrial revolution, humans have been exploiting the planet's natural resources in
Biotechnology an unsustainable manner. This has caused some irreversible impacts on the environment. Because of this, we are facing a
Green chemistry change of philosophy within the scientific community about chemical and industrial processes. During the 1990s, the concepts
Biofuels of green chemistry began to solidify, while in parallel some major advances in biotechnology were achieved through
Biopolymers
developments of genomic sciences and genetic and metabolic engineering. This work will discuss some new insights into the
Organic synthesis
use of biotechnology as an important tool in green chemistry, showing new applications to biopolymers, biofuels and as a new
alternative to tradi-tional organic synthesis, making chemical processes more sustainable and less damaging to the planet.

1. Background 2009). After all, as proposed by the classic thought of A.L Lavoisier:
"Nothing is lost, nothing is created, everything is transformed." There-fore,
Since the mid-eighteenth century with the first industrial revolu-tion, the industrial waste will continue to "exist" on this planet as long as a better
mankind has been changing the way of extracting resources from the planet. destination is not given to it, because the natural processes of degradation are
Initially, we implemented a basically artisanal production system, in which much slower than that of the producing new waste. Moreover, most of these
humans themselves were the main production tools (de Vries, 1994). This was advances depend on energy derived from fossil fuels, which besides
drastically altered by the introduction of machinery, driven by energy derived contributing to global warming and other en-vironmental problems are not
from fossil fuels such as coal (Fahrenkamp-Uppenbrink, 2015; Berg, 1978), renewable (at least not in the "short" run).
mainly in the textile and metallurgical industries, which as a result expanded
dramatically (Fores, 1981). At the end of the nineteenth century, the world As known, pollution has caused much irreversible damage to the
experi-enced the second industrial revolution, in which rapid technological environment. The increase in the anthropogenic emission of greenhouse gases
development led to most of the industrial advances that allowed the sale of (GHG)1 from burning fossil fuels and forest destruction is now a global
consumer goods that still are important to the society today. This brought the concern that demands a unified effort from all nations, with the aim of slowing
establishment of the automotive and aviation industries, production of oil- global climate change (Allison and Bassett, 2015). The rapid industrialization
derived materials, the pharmaceutical industry, and computing (the base for processes has also caused damage in other spheres, such as soils and water,
the ongoing information revolution) (Spilhaus, 1970). impacting in multiple ways the life quality of all living beings (Kelly and
Fussell, 2015; Grossman and Krueger, 1995).

However, the unsustainable exploitation of Earth's natural resources in the In the twenty-first century, although belatedly, society in general and the
last 300 years has obviously caused impacts that threaten the well-being of scientific-industrial community now largely agree that the ways of interacting
future generations. The waste generated as a consequence of all the industrial with the environment and exploiting resources need to be changed. In the
advances of recent centuries is not adequately treated or sufficiently recycled, midst of the third industrial revolution, characterized by an extremely refined
posing one of the greatest challenges to society (Giusti, 2009; El-Fadel et al., and advanced chemical industry, we are also facing a new revolution that
1997; Houk, 1992; Robinson, comes from the incredible advances of the

E-mail address: [email protected].

1 Green house gases abrangem CO2, N2O, CH4 e gases fluorados.

http://dx.doi.org/10.1016/j.scp.2017.09.002
Received 23 March 2017; Received in revised form 27 September 2017; Accepted 28 September 2017
Available online 13 October 2017
2352-5541/ © 2017 Elsevier B.V. All rights reserved.
M. Fasciotti Sustainable Chemistry and Pharmacy 6 (2017) 82–89

genomic sciences (Abelson, 1998). The general hope is that this current as an industrial waste, and some microorganism or enzyme capable of
revolution can address the impacts generated by the previous un-sustainable transforming it into the desired final material or product. In practice, this
revolutions. process offers advantages to traditional methods, by improving the selectivity
of chemical processes. In addition, genetic engineering offers new pathways
to obtain desired products, sometimes with lower pro-duction costs and using
2. Important concepts: green chemistry, biotechnology and white
safer and more sustainable materials.
biotechnology
During recent decades, biotechnology has been applied in several areas of
medicine, for the development of new pharmaceutical drugs (Olsen et al.,
With the change in the mentality towards the planet's serious en-
2004; Drews and Ryser, 1997; Orive et al., 2003; Butcher et al., 2004),
vironmental problems and the general acceptance of the fact that something
vaccines and antibodies (William, 2005; Luckow and Summers, 1988), and
needed to be done to mitigate the damage, the concept of "green chemistry"
medical materials for transplants or prostheses (Yang et al., 2014; Gaharwar
was developed, which basically encourages a closer look at the impacts of
industrial processes on nature. et al., 2014; Ding et al., 2014; Fu et al., 2013; Hench and Polak, 2002); in the
area of food and agriculture, to produce eco-friendly food (Andrés et al.,
The first principle of the United Nations Conference on Environment and
2016; Alam et al., 2014; Kasmi et al., 2017); and for environmental
Development, which met in Rio de Janeiro from 3 to 14 June 1992, states
protection, with the development of new biofuels derived from plants and
that:
other renewable sources (Peralta-Yahya et al., 2012; Georgianna and
“Human beings are at the center of concerns for sustainable development. Mayfield, 2012), among other applications. In the early 2000s, more than 120
They are entitled to a healthy and productive life in harmony with nature.” products obtained through biotechnology processes had been approved by the
FDA (U.S. Food and Drug Administration) (https://www.bio.org/media/press-
re-lease/biotechnology-fundamentals-solid, 2016).
The role of chemistry to achieve this goal is undeniable. According to
data from the United States Environmental Protection Agency (US-EPA), In this way, biotechnology is a science that can be applied as a tool to
around 278 million tons of toxic wastes were generated in 1991, of which the improve processes in virtually unlimited applications. It may be the key
greatest portion came from the chemicals industry (Sanders, 2011). As a science by making traditional chemical processes into "green" processes,
result, the biggest corporations, such as Dow Chemical and DuPont, have where the combination of biotechnology and green chem-istry leads to so
begun to devote efforts to comply with increasingly restrictive environmental called "white biotechnology". It is an area that has emerged rapidly in recent
regulations, thereby tapping into green chemistry practices. This term, which decades and refers to the use of biotechno-logical approaches in the
was proposed by the che-mist Paul Anastas in the 1990s, basically aims to laboratory and industrial production of fine chemicals, biofuels and
develop and imple-ment chemical products and processes to reduce or agricultural products, among many others (Gupta and Raghava, 2007). The
eliminate both the use and generation of substances harmful to human health main focus of white biotechnology is the development of "clean" processes
and the environment, by applying the twelve basic principles of green chem- that result in reduction of green-house gas emissions, water and energy
istry, already well known (Anastas, 1998). consumption, and generation of industrial waste. It is a truly multidisciplinary
frontier area, which closely links the main areas of chemistry and biology.
In practice, these concepts of green chemistry have revolutionized the
way of planning new projects and research in chemistry and have impacted its On the other hand, major challenges still exist when combining these two
sub-areas in the most diverse applications (Horváth and Anastas, 2007; principles (biotechnology and green chemistry) still running around the
Fantke et al., 2015). Moreover, in parallel with this change of mentality, there upstream and downstream processes: from early cell culti-vation, raw
have been significant advances in bio-technology, which - under some materials supply, to the final challenge to obtain the target material with the
circumstances – can provide valuable or alternative tools for the desired characteristics, after separation and pur-ification procedures. All these
implementation of some of the 12 concepts of green chemistry. steps must be critically evaluated, be-cause they can be intensively
demanding of energy, chemicals and water. Irrespective of whether a
Therefore, biotechnology is a general term used over a very broad field of genetically engineered strain is used, both processes must be considered as
study. According to the Convention on Biological Diversity, biotechnology they can make a biotechnological process unsustainable, and not green at all
means (Zaid et al., 1999): (Chmielowski et al., 2007; Junker et al., 2004).
"Any technological application that uses biological systems, living or-
This work will emphasize some examples of applications of bio-
ganisms, or derivatives thereof, to make or modify products or processes
technology in green chemistry, also discussing some important points related
for specific use."
to its true greenness.
It is an interdisciplinary area of science in which the main focus is
studying the use of cells, microorganisms, in vitro or in vivo, for the 3. Applications
production of new products through new processes. It is also an area of
science that has been applied for centuries: a simple fermentative process for 3.1. Biotechnology applied to materials: biopolymers
the production of alcoholic beverages can be considered a biotechnological
process, since it uses microorganisms to transform sugar into alcohol. The petrochemical industry is one of the major sources of air pol-lution
However, the main breakthrough in biotechnology was the provided by and the increase in GHG emissions (https://www3.epa.gov/cli-
significant advances in the genomic sciences (Cañestro et al., 2007), and of matechange/ghgemissions/). The manufacture of petroleum-based polymers
genetic and metabolic engineering (Parekh, 2004). plays a significant role in generating this pollution (Fu et al., 2003). In
addition, their high durability and strength, in the past seen as “good
The genomic sciences have had a great impact on the chemical in-dustry, qualities”, are now a serious environmental problem, since these polymers
since it is now possible to genetically modify microorganisms to target certain accumulate in the environment, and hundreds of years are required to
metabolic behavior according to what one wishes to obtain. This is called completely decompose them, during which they often re-lease toxic
metabolic engineering and these genetically modified microorganisms are substances that can contaminate soil and groundwater (Webb et al., 2013;
known as "engineered" microorganisms (Parekh, 2004). Thus, instead of Alay et al., 2016). Biodegradable polymers are a sustainable alternative, since
thinking about a longstanding che-mical process that has reagents, catalysts, by definition they are degraded through enzymatic actions that tend to
products and byproducts, the chemical process is designed to use some kind continuously decrease their molecular masses. The term "degradable" is used
of starting material, such when hydrolysis or enzymatic

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degradation occurs, or when the polymer chain cleavage processes occurring cycle, which involves pentose phosphate, by cloning genes from Cau-lobacter
during degradation are not fully known. crescentus. These cells were then engineered to simultaneously use glucose
Biopolymers are polymer chains synthesized by living organisms and are and xylose to produce the respective lactic and glycolic acids. Third, the
therefore perfect examples of the application of biotechnology in green enzyme propionyl-CoA transferase (Pct) was also en-gineered to convert in
chemistry, due to several aspects: 1) because they are biode-gradable, they vivo the glycolates and lactates to their CoA-derived intermediates. Finally,
reduce the accumulation of industrial waste; 2) they tend to generate less another enzyme involved in the synth-esis of polyhydroxyalkanoates (PHA),
pollutants during production; 3) they can use waste or byproducts as starting an engineered PHA synthase, was used to copolymerize the CoA derivatives
material; 4) they can be less toxic, which in-creases the possibilities of and form the PLGA. The authors then used the same approach, i.e., the novel
application in medicine and food, for ex-ample; and 5) they tend to metabolic pathway of the engineered E. coli strain resulting from these
biodegrade forming innocuous (or less toxic) products to the environment. various gene in-troduction/exclusion steps, to verify whether it would be
Some examples of natural biopolymers are polysaccharides including starch, possible to synthesize other copolymers with other monomers having different
chitin/chitosan, cellulose, algi-nate and other carbohydrate polymers. Among compositions. For this, in addition to glycolic acid, they used other chain
polymers formed from animal proteins are gelatin, wool, silk, collagen and acids, such as 2-, 3-, 4-, 5-, and 6-hydroxycarboxylic acids as starting models
gluten (Thakur et al., 2014). .

The production of biopolymers is an extremely broad subject, especially This work is a very illustrative case study of the massive application of
those produced by bacteria and those derived from biomass. The use of genetic engineering for the synthesis of biopolymers. For example, to
bacteria for the production of biopolymers has opened a wide range of introduce the Dahms cycle in the metabolism of E. coli, the authors cloned
possibilities for the production of different materials, since bacteria are able to two genes from another microorganism (C. crescentus), xylB and XylC,
efficiently convert different carbon sources into several types of polymers with responsible for decoding xylosedehydrogenase and xylonolacto-nase. The E.
different chemical and mechanical properties. Most bacterial species coli strain containing this new plasmid3 was able to pro-duce 0.89 g per liter
synthesize polymers in extracellular form (Rehm and Valla, 1997). Most of of glycolic acid from xylose as the only carbon source.
these polymers are formed from inorganic polysaccharides, polyesters,
polyamides and polyanhydrides such as polyphosphates (Pham et al., 2004). The characteristics of the produced PLGA were also comparable to the
Various biopolymers ob-tained through bacteria are already commercially PLGAs chemically synthesized with respect to the monomeric composition
produced on a large scale, for example xanthan and dextran polysaccharides. and molecular weight (15,000–25,000 Da), besides having the same thermal
In 2010, Rehm and co-workers published a review in Nature Reviews and plastic properties. However, in the chemical route for producing PLGA,
Microbiology on the most important aspects of the production of biopolymers metal catalysts are generally used, and the removal of the byproducts adds
by bac-teria (Rehm, 2010). costs to the process. Removal of these catalysts and byproducts is extremely
important for PLGAs used in the medical field, since they will come into
One of the most important applications of biopolymers is in the medical contact with biological fluids and tissues and therefore need to be safe. The
area. Delplace and Nicolas (2015) reviewed several methods of rendering authors stated that this is not as problematic in the metabolic pathway for
vinyl polymers degradable for medical applications. Parti-cularly in medicine, PLGA production re-ported in the article. In addition, the approaches used
polymer degradability is extremely important for the choice of materials for a could be applied to several other biopolymers that can be made from
given application, since biopolymers can be used for the transport of slow renewable sources through engineered microorganisms. It is expected that this
release drugs in the body, as well as for synthetic fabrics and medical area will continue to develop during the next years (Zhu et al., 2016).
implants. For each application, the bio-polymer must be compatible with the
biological homeostasis of the human body and degrade with a velocity that is Other biopolymers that are receiving considerable attention are those
compatible with the function it will perform within the organism. It is difficult derived from lignocellulosic biomass, since this material is an extremely
to imagine this level of optimization of these materials without the advent of abundant renewable carbon source and does not have a di-rect application
biotechnology-related practices and principles. other than burning for the generation of energy. Lignin and cellulose are
generally byproducts generated in industrial processes such as paper, sugar
To illustrate this , a recent paper published in Nature Biotechnology and ethanol production. They are natural polymers with many possibilities for
described a new one-step method for the production of carbohydrate conversion into new biopolymers to replace those derived from petroleum
polylactate-co-glycollate (PLGA) through fermentation by engineered (Fernandes et al., 2013).
Escherichia coli, from various stages of genetic modification, to enable The biopolymers that have the greatest potential to replace syn-thetic
polymerization of D-lactyl-CoA and glycol-CoA in PLGA from xylose and products of petrochemical origin are the bioplastics, with appli-cation in the
glucose (Choi et al., 2016). packaging segment, as they can be recycled as well as being biodegradable
PLGA is a random copolymer composed of lactic and glycolic acids. It (Soroudi and Jakubowicz, 2013). Polyethylene (PE) made from ethanol from
deserves special attention because it is one of the few commercially available sugarcane is now being used in several countries, where it is called "I'm
biopolymers that have been approved by the FDA, being biodegradable, Green" or “Plant bottle”, as a reference to its green origin
biocompatible and non-toxic. It is applied primarily in the medical field. The (http://www.braskem.com.br/Portal/Principal/
classic synthesis of PLGA is through a chemical route in which lactic and Arquivos/Download/Upload/Catalogo_PE_Verde.pdf). However, in terms of
glycolic acid dimers are copolymerized in the presence of organometallic resource efficiency, ethanol production still waste carbon, since half of carbon
catalysts. The idea of this article was to engineer a strain of Escherichia coli is lost as CO2, making it not ideally sustainable.
(E. coli) so that this copolymeriza-tion was performed in only one process, The better bioplastics will be those that can be efficiently produced from
through fermentative meta-bolism. biomass and recycled even when mixed with other materials (Hassan et al.,
2013). Without efficient production and recycling bio-technological processes
To make PLGA directly from carbohydrate fermentation, initially E. coli will not succeed. Moreover, the ethanol
was engineered to produce glycolate from glucose by introducing the Dahms
cycle2 into the bacterial metabolism, rather than the native
(footnote continued)
resulting in 2-keto-3-deoxyxylonate, which is then cleaved by an aldolase to form pyr-uvate and
glycoaldehyde.
2 3
The Dahms Cycle is an oxidative route in which D-xylose is oxidized to D-xylyl lactone by Plasmids are double circular DNA molecules capable of reproducing independently of
a D-xylose dehydrogenase, followed by a lactonase to hydrolyze the lactone to d-xylic acid. chromosomal DNA, used to multiply specific genes. The gene to be replicated is inserted into a
Then, through the action of xyloneate dehydratase, a water molecule is removed, plasmid and then inserted into the microorganism.

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M. Fasciotti Sustainable Chemistry and Pharmacy 6 (2017) 82–89

produced by fermenting sugarcane still generates residues, mainly ba-gasse, widely produced worldwide because of their greater ease of production, such
which in this case can be a good source of carbon for the pro-duction of as biodiesel from soybean, sunflower, corn, canola and re-used vegetable oils,
biopolymers. Ethanol from sugarcane is already widely used in automotive or animal fats. Biodiesel production in the United States was about 105
fuels (it is the main biofuel used in Brazil). Thus, Shi and Chen published a million gallons (about 397 million liters) in February 2016
paper in Sustainable Chemistry and Engineering (Chen and Shi, 2015), (http://www.eia.gov/biofuels/biodiesel/production/). Bioe-thanol, however, is
aiming at the direct transformation of sugarcane ba-gasse into bioplastics by mainly obtained from corn, sugarcane, wheat and beet (Nigam and Singh,
modifying it through the use of phthalic an-hydride (PA). They performed 2011). The United States and Brazil are the world's largest producers of
homogeneous esterification of cane ba-gasse with phthalic anhydride using 1- bioethanol, which is mostly obtained from sugarcane in Brazil and from corn
allyl-3-methylthiimidazolium chloride (AMIMCI) – an ionic liquid – as in the U.S. In 2015 in Brazil, more than 10 billion liters of sugarcane
solvent. Bagasse transforma-tion was obtained in a more chemical than bioethanol (http:) was consumed.
biological way - which would be done through microorganisms – but it still Second-generation biofuels generally use lignocellulosic residues from
poses a problem related to green chemistry, that is, to recycle one of the most agriculture or the paper industry, which can be converted into various types of
important wastes from a biotechnological process (sugarcane fermentation). liquid or gaseous fuels through thermochemical, en-zymatic, pyrolytic and
other processes (Balat et al., 2008; Mood et al., 2013). Examples are the
Sugarcane bagasse is a natural composite of lignin, hemicellulose and bioethanol obtained through lignocellulosic biomass and the diesel obtained
cellulose, with several hydroxyls in its structure, which can be chemically by the Fischer-Tropsch process (Stöcker, 2008; Leckel, 2009; van Vliet et al.,
modified to alter its properties. However, the complexity of this biomass 2009).
makes research seeking the direct transformation of this material less Third-generation biofuels have recently been developed, involving the use
common and more challenging. The authors reported that these few studies of algae. Initially, biofuels made from algae were defined as second-
were not very successful, because the formed bio-plastics were fragile. The generation, but when it became clear that algae were able to provide much
authors (Chen and Shi, 2015) concluded that the addition of the phthalic better yields than processes with other biomasses and through theoretically
group acts as a plasticizer for sugarcane bagasse, but that improvements still simpler production steps, algae were included in a new category. They have
need to be made to improve the flexibility of this biopolymer without been the subject of massive research for ef-ficient production of biofuels
reducing its tensile strength. (Scott et al., 2010). A very interesting advantage of using algae as biomass is
Nevertheless, before planning a new biopolymer, some important aspects that they can be grown in wastewater such as sewage, acting simultaneously
should be taken into account: What plastics really need to be replaced , that is, for bioremediation (Kligerman and Bouwer, 2015).
what are the principal fossil fuel-based polymers that are causing relevant
damages to the planet? If wanting to replace a non-biodegradable fossil-fuel Fourth-generation fuels, which are the state-of-the-art now, are defined as
polymer, how much of the biobased one is needed to replace the fossil-based photobiological fuels or "electrofuels," and emerge from the incredible
ones? And finally, are sufficient feedstocks (biomass, raw materials, waste and advances in biotechnology and metabolic engineering (Aro, 2016). Both
byproducts) available to produce the biopolymer for replacement? If these third- and fourth-generation fuels use photosynthetic mi-croorganisms to
answers tend to be positive, then a greener scenario can be expected (Yu and produce fuels: in the third generation, the way to do this is basically to
Chen, 2008). process the algal biomass to extract the desired biofuel from it, whereas in the
fourth generation metabolic engineering is applied to algae, specially
3.2. Biofuels cyanobacteria and yeast, for the produc-tion of photosynthetic fuels
(Bharathiraja et al., 2015). In practice, the desire is to create a kind of "self-
Currently, the energy mix in the world is still based on petroleum sustaining biorefinery" in which algae are capable of producing biofuels or
derivatives such as diesel, gasoline and aviation naphtha (IEA, 2007). any chemicals only from CO2 and electromagnetic energy from sunlight, a
However, it is widely argued that the uncertainty of the future avail-ability of process also called solar-to-fuel, when the final product can be used as a fuel
oil, the fluctuations of its value in the international market, and mainly the directly (Tuller, 2017; Listorti et al., 2009).
pollution generated by burning fossil fuels, requires finding other carbon
sources to replace it. Production of fuels derived from renewable sources of Fu and co-workers published a review in 2011 in Energy &
carbon is one of the main areas of applying biotechnology. Today there is Environmental Science (Lu et al., 2011) summarizing the lit-erature to that
widespread research to find new re-newable sources and develop new date on the production of biofuels through algae, dis-cussing their genetic
processes to make various biofuels. manipulation for metabolic optimization. They discussed various species of
All biofuels have the same conceptual basis: they are produced through algae from the genomic point of view and described case studies of the
sources that convert solar energy into chemical energy by capturing CO2 production of biofuels. Currently, there are studies and applications for the
through photosynthesis (Sarkar and Shimizu, 2015) to produce a series of production of: (i) biohydrogen by algae through solar energy and water
compounds derived from their own metabolic pathways. However, the (Kruse and Hankamer, 2010); (ii) bioisoprene (2-methyl-1,3-butadiene),
sustainability or greenness of a biofuel can be controversial. If one consider generated from the fixation of CO2 through the mevalonic acid route,
aspects such as how much fertilizer, water and soil will be required to incorporated into bacteria through the cloning of plant genes (Yang et al.,
produce a specific raw material for biofuel; or how efficiently the biomass will 2012); (iii) bioethanol, mainly by cyanobacteria, but also through metabolic
be converted into biofuel; and finally how much greenhouse gases emissions engineering that integrates genes related, for example, to alcohol
are produced from seed to fuel, one can conclude that some biofuels do not dehydrogenases (ADH) and pyruvate decarboxylases (PDC), allowing yields
satisfy the principles of green chemistry at all (Scharlemann and Laurance, comparable to the classic microorganism of alcoholic production, the famous
2008). Sacchar-omyces cerevisiae (John et al., 2011; Kim et al., 2011); (iv) butanol
Biofuels are currently classified as first, second, and most recently third and other four-carbon alcohols, by Clostridium acetobylicium and Synecho-
and fourth generation (Cherubini, 2010). The first generation is produced coccus elongatus, which after being engineered are used to produce
through raw materials that generally compete with the food industry, such as isobutyraldehyde, isobutanol and butanol directly from CO2 (Astumi et al.,
edible oilseeds, sugarcane, corn, beet and wheat (Gnansounou and Dauriat, 2008; Atsumi et al., 2009); and (v) biodiesel, the most promising biofuel that
2005). Because of this competition, these biofuels result in both ethical and can be produced through algae (Beer et al., 2009; Mata et al., 2010; Chisti,
political dilemmas, and in addition they still generate waste from their 2008). Some oily algae have 30–60% (w/w) of triacylglycerols on a dry basis
processing. To overcome these is-sues, the production of second generation and are synthesized for lipid accumula-tion in their chloroplasts. In order to
biofuels targets the use of raw materials mainly from agricultural waste or obtain better yields in the pro-duction of biodiesel precursor lipids such as
non-edible biomass sources (Sarkar et al., 2012). However, first-generation triacylglycerols, the
biofuels are the most

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microalgal species are evaluated in different culture media, containing to the higher melting point of these fatty acid esters. However, C14 fatty
different nutrients and physico-chemical conditions such as pH and acids, ideal for biodiesel performance, were not found in the species that
temperature, since these parameters have a strong influence on the produced the highest total fatty acid yield. Some could may serve as models
composition of the biomass, because algae tend to adapt their meta-bolism to for genetic engineering, since they presented the highest concentrations of
the growth conditions related to their culture medium. C14 (but not the highest total fatty acid productivities), such as Prymnesium
In a recent study, Nielsen and co-workers (Kerkhoven et al., 2016) parvum, which had 28% C14, and Cylindrotheca sp., which reached up to
showed that under growth conditions with limited nitrogen supple-mentation, 30%. In the end of the study, the authors summarized in tables all values
the regulation of amino acid metabolism controls the flow of lipid determined for each analyzed parameter and categorized the best species for
accumulation in the cells of this microorganism, increasing the concentration each biotechnological application, making the article an important source of
of this class of molecules. They studied Yarrowia lipoly-tica, which is an information on the biotechnology of algal biomass.
oleaginous yeast able to accumulate up to 70% of total lipids in its biomass,
with potential for the production of biofuel and other applications in From the biotechnological point of view, first-generation biofuels use less
biotechnology (Gonçalves et al., 2014). Briefly, they cultivated Y. lipolytica sophisticated, or more trivial, biotechnology tools. The pro-duction of
under two conditions: one with restriction of carbon (glucose) and another biodiesel through the transesterification of oils is essentially a chemical
with restriction of nitrogen (in the form of ammonia) in the culture medium. process, but when recalling that many oilseeds have un-dergone several stages
After chemical analyses (by chromatographic and mass spectrometric of genetic improvement to develop cultivars that have better yield for the
techniques) of some classes of lipids, they concluded that the nitrogen production of biofuels, it is possible to see clearly the role of biotechnology
restricted condition favored higher production of lipids, mainly ergosterol, (Divakara et al., 2010). In this respect, obtaining ethanol from alcoholic
phosphatidylcholine and triacylglycerols. They then extracted and analyzed fermentation of sugars is one of the oldest biotechnological processes used. In
the RNA and verified the changes in gene expression and changes in fact, second-generation fuels increasingly use more advanced biotechnology
transcription that were responsible for the synthesis of lipids under conditions tools, which increase in complexity according to the generation of biofuel
of nitrogen limitation. This study is important because understanding the (Keasling and Chou, 2008).
metabolic pathways involved in lipid synthesis, as well as the genes
responsible for these changes in microorganisms, is necessary for the study
with other microorganisms or for cloning for genetic improvement and me-
tabolic optimization. 3.3. Organic synthesis

Also regarding third-generation biofuels, an article published in Nature Organic reactions are the center of synthesis and processes of
Scientific Reports by Slocombe et al. (2015) presented data ob-tained through transforming matter. When conducting a holistic analysis, all processes and
a comprehensive and systematic analysis of the biomass composition of applications already mentioned in this text involve organic reac-tions of
different species of algae. Based on these results, the authors discussed the different complexities and for different purposes. Organic synthesis is the
best potential biotechnology applications: the production of biofuels, biogas, central science behind most of today's industrial pro-cesses and thus is also an
bioremediation, food, and so on. To date, this may be the most comprehensive important source of pollutants and toxic wastes. Therefore, the green
study of the characterization of algal biomass. The main objective of the work chemistry idea calls for changing the way organic synthesis is done today by
was to identify the genera, species and strains of algae suitable for large-scale developing processes that obey green principles (Bevan and Franssen, 2006).
production in industrial biotechnological processes, such as the production of The ideology of green chemistry generally demands that syntheses be sought
biodiesel, biogas, or nutraceutical foods with high nutritional value. The that have greater energy efficiency, selectivity, simplicity, and safety for
authors’ goal was to obtain strains capable of growing satisfactorily in salty nature and human health (Li and Trost, 2008).
water, without the need for additional nutrients such as phos-phates and
nitrates. This would result in a drastic reduction in costs for large-scale The main characteristics of a green organic synthesis are: (i) economy of
production. For that, they defined a standardized culture medium for the atoms, in which the greatest possible amount of reagents must be incorporated
growth of all strains evaluated, which simulated the physical and chemical in the product (Trost, 1995); (ii) direct conversion of C–H bonds to C–C
conditions of seawater. Thus, it is important to perform a robust comparative bonds (Trusova et al., 2016); (iii) no use of pro-tective groups, since this
analysis of the composition of all biomass sources, since the culture medium increases the number of steps in the synthesis (Young and Baran, 2009); (iv)
has strong influence on the pro-ductivity of certain classes of metabolites, as cascade reactions which can incorporate a series of steps for the synthesis of
previously discussed and exemplified in the work of Nielsen et al. (Chisti, the product in only one process (Webb and Jamison, 2010); (v) the use of
2008). The authors evaluated 175 algal species, cataloged in CCAP (Culture biocatalysts, which are more selective and efficient, while at the same time
Collection of Algae and Protozoa, UK), suitable for long growing times. being non-toxic (Reetz, 2013); and (vi) use of less toxic solvents such as
water, supercritical CO2 and ionic liquids (Sheldon, 2005).

Specifically for biodiesel production, the best species/strains were those of In this way, the already defined "white biotechnology" has great
Nannochloropsis oceanica CCAP 849/10 and Chlorella vulgaris CCAP importance within organic synthesis. In many cases, the use of bioca-talysts
211/21A. However, the amount of polyunsaturated fatty acids (PUFA) found as enzymes or microorganisms can be a better alternative route to traditional
in these species/strains can negatively impact the stabi-lity of biodiesel due to synthesis processes, generally allowing greater selectivity and yield (Reetz et
the degree of unsaturation. In general, the de-termined concentrations of al., 1997) and in most cases being compatible with the use of aqueous media.
PUFA were higher than those observed for the usual raw materials employed In addition, the advances in metagenomics4 have allowed the production of
for biodiesel production, with a mean of 34%, but varying from 4% to 74% extremely specific enzymes that cannot be obtained through culturing
among all species analyzed. This value was 32% in C. vulgaris, but restricted microorganisms (Uchiyama and Miyazaki, 2009).
to three unsaturations per chain at most.
The applications of enzymes and microorganisms as alternative synthetic
All species of Nanochloropsis were found to have low concentrations of routes are a topic apart, due to the numerous applications that
PUFAs (5–11%) and half was eicosapentaenoic acid (C20:5). Due to the high
concentrations of C16: 0 and C16: 1 acids in N. oceanic species, it was the
4
one that presented fatty acids with the shortest chains on average among the Metagenomics is the genomic analysis of the community of microorganisms of a given
environment by independent cultivation techniques. This approach consists of extracting DNA
analyzed species. This can be seen as an advantage, since acids with long directly from the environment and constructing a metagenomic library with this mixed genome.
chains cause cold starting problems in engines due

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have been developed, mainly for the synthesis of chiral compounds and biocompatible. It also reduces the costs of their production, since all the
amino acids (Nestl et al., 2014). An interesting example that serves as a case chemical and technological refinement for the synthesis of semi-conductors is
study of organic synthesis performed through microorganisms was published not necessary in this case. Living organisms have the endogenous ability to
in 2013 by Jiang and co-workers in Biosource Technology (Jiang et al., perform the synthesis of inorganic materials, such as nanomaterials. One
2013). They proposed the synthesis of succinic acid through Actinobacillus advantage of this is that they generally can very precisely control the shape
succinigeses. The biotechnological synthesis of succinic acid has been a and crystallinity of a developing inorganic material. In this respect, Iverson
recurrent focus of research, and there are several methods for obtaining it and co-workers published the first report of semiconductor nanocrystal
through microorganisms. These studies were reviewed and published in synthesis in bacteria in 2004 (Sweeney et al., 2004). They showed that E. coli,
Current Opinion in Biotechnology by Lee et al. (Ahn et al., 2016). when incubated with cadmium chloride and sodium sulfide, synthesized
intracellular cad-mium sulfide (CdS) nanocrystals. The CdS nanoparticles
The reasons for large number of studies of succinic acid is the fact that it were appar-ently being formed inside bacterial cells following transport of
is an organic molecule with strong industrial interest, since it serves as a Cd2+ and S2- ions, and this formation is strongly dependent on the bacterial
precursor for several other products such as tetrahydrofuran, γ-butyrolactone, growth phase and strain.
1,4-butanediol, and others (Zeikus et al., 1999). In-dustrially, synthesis of
succinic acid is accomplished by the hydro-genation of maleic acid, oxidation The precipitation of semi conductive nanoparticles is mainly per-formed
of 1,4-butanediol alcohol and car-bonylation of ethylene glycol (Cornils and by photosynthetic microorganisms. However, the authors of the solar-to-
Lappe, 2005). chemical approach (Sakimoto et al., 2016) argued that their metabolic
The objective of Jiang et al. (Nestl et al., 2014) was to obtain suc-cinic pathways are sometimes less interesting than the metabolic pathways of non-
acid through cellobiose obtained through enzymatic hydrolysis (enzymolysis) photosynthetic microorganisms, because they offer simpler ways of obtaining
of sugarcane bagasse by the metabolism of Actinobacillus succinogenes, a larger diversity of products through the simple reduction of CO2 to new
which is a gram negative bacterium found in bovine rumen and is one of the molecules. In addition, there is a thermodynamic advantage (Fast and
most promising bacteria for the production of succinic acid through sugar Papoutsakis, 2012) of photo-sensitizing non-photosynthetic bacteria to reduce
sources. Therefore, several production routes through a fermentative process CO2 to other organic compounds, caused principally by the metabolic
have been proposed using in-dustrial and agricultural wastes as starting mechanisms present in these bacteria, such as the called Wood-Ljungdahl
material for organic synth-esis, since they are renewable sources of carbon pathway.5 In this mechanism, CO2 is reduced to acetyl-CoA, which is a fairly
(Bevilaqua et al., 2015). Therefore, this work presented a biotechnological common biosynthetic intermediate – and possibly to acetic acid – both of
route to ob-tain succinic acid, using sugars obtained through the hydrolysis of which can be biotransformed into other more complex organic molecules
su-garcane bagasse, making this synthesis renewable and in line with several through the metabolism of engineered microorganisms.
concepts of green chemistry.
As previously mentioned, the authors then developed a hybrid system
Another example of alternative synthesis development is the so-called containing Moorella thermoacetica, a non-photosynthetic bac-teria reducing
solar-to-chemical synthesis via microorganisms. In this chemical route, a CO2, and CdS as biologically precipitated semiconductor nanoparticles. The
microorganism with photosynthetic behavior – native or arti-ficially photosynthesis of acetic acid by M. thermoacetica and induced by the CdS is
introduced to its biological system – is used in the high-effi-ciency synthesis a process in a pot consisting of two stages: first, the precipitation of CdS by
of several compounds, using CO2 as the carbon source (Woo, 2017). M. thermoacetica is triggered by the addition of Cd2+ ions (by the addition of
Cd(NO3)2 to the culture medium containing glucose during exponential cell
An interesting example of the implementation of such a system was
growth), supple-mented with cysteine, which acts as a source of sulfur. M.
published in Science by Yang and co-workers in 2016 (Sakimoto et al., 2016). thermoacetica uses the electrons generated photochemically by light
The conversion of solar energy into chemicals has a similar scheme to that of irradiation in the CdS to perform the photosynthesis. The absorption of a
the fourth-generation fuels mentioned in the previous topic. But in this work, photon by the CdS produces a vacancy and a pair of electrons, which are
the authors developed a hybrid system that combined the high light capture introduced in the Wood-Ljungdahl cycle to synthesize the acetic acid from
efficiency of inorganic semiconductors with the ability of selective and CO2. Cysteine neutralizes the vacancy generated in the semiconductor,
efficient synthesis of biocatalyst mi-croorganisms. They induced the forming its oxidized disulfide, cystine (CySS). All the experimental
photosensitization in an acetogenic non-photosynthetic bacterium, Moorella procedures - including the quantities of culture media, M. thermoacetica and
thermoacetica, through the biological precipitation of cadmium sulfide
Cd2+ ions required to trigger the CdS nanoparticle synthesis, were explained
nanoparticles that ag-gregate to the bacterial cells, resulting in the
by the authors in details in the supplementary materials of their manuscript.
photosynthetic synthesis of acetic acid from CO2, through the metabolism of These parameters are of great importance and need to be critically evaluated
this microorganism. Although it is a trivial molecule, the approach of this to consider solar-to-chemical synthesis a sus-tainable process.
work has great relevance and innovation, since it can be used for the synthesis
of other more complex molecules in future studies.
Moreover, to further characterize the photosynthetic mechanism induced
The authors emphasized that there is a need to improve the capture of in M. thermoacetica, they irradiated the culture by simulating sunlight for 12
solar energy for sustainable chemical synthesis, and that some major advances hours cycles, as would normally be the case in circadian cycles. They
have been made with the development of solid-state inorganic observed that, curiously, the production of acetic acid did not cease, and
semiconductors, which generally do this more efficiently than photo-synthetic actually increased, even in periods of no light. The ex-planation given by the
processes. However, they do not have the same performance in transforming authors is possible accumulation of biosynthetic intermediates during periods
this energy into new carbon bonds (mainly in the conversion of CO 2 into of illumination, which are consumed during periods of dark. This shows that
more complex molecules), that is, forming new C–C bonds for organic the process could be viable under natural lighting conditions. In addition,
synthesis, something that photosynthetic systems do with high efficiency. during the natural pho-tosynthesis of photosynthetic organisms, metabolic
Therefore, the approach used in their work aimed to combine the advantages alteration occurs in the absence of light, which was not observed in the
of these two mechanisms: the ability to capture solar energy of photosynthetic
semiconductors and the high catalytic per-formance of biological systems.

One interesting aspect of this paper is the use of microorganisms to 5

In the Wood-Ljungdahl pathway, also known as the acetyl-CoA reduction pathway, some
induce the precipitation of semiconductor nanoparticles. This approach has
microorganisms use hydrogen as an electron donor and CO2 as an acceptor for biosynthesis.
some advantages because the semiconductors are, therefore,

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M. Fasciotti Sustainable Chemistry and Pharmacy 6 (2017) 82–89

mechanism used in this study. the environment and human health. Therefore, some improvement by using
That work showed that the use of hybrid systems of naturally pre-cipitated more efficient and non-toxic semiconductor nanoparticles needs to be
semiconductors and photosensitized bacteria can be an effi-cient and self- achieved. But arguably this may be one of the most sustainable synthetic
sustaining alternative for the synthesis of various che-micals, even using routes developed.
engineered microorganisms in future studies, for a more diversified To sum up, research is being conducted on many fronts and will certainly
production of other more organic molecules obtained by reducing CO2. lead to important discoveries to mitigate the damage caused by current
unsustainable processes, through the combination of "green" chemistry and
applied biotechnology.
4. Critical view and future perspectives
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